The technique of small fragment homologous replacement (SFHR) is of particular interest in gene therapy as it has potential for treatment of inherited genetic disorders, such as cystic fibrosis. In such patients, mutant genomic sequences would be corrected by their exchange with small fragments of wild-type homologous DNA. However, before SFHR can be used in clinical trials, further research is necessary. The proposed project is to develop in vitro systems that can be used to study the effectiveness and safety of SFHR. Expression vectors containing a mutated gene for one of three reporter proteins (Beta-galactosidase, neomycin resistance, green fluorescent protein) would first be constructed and then stably integrated into the genome of transformed human epithelial cells. SFHR would then be performed on these cells. Successful replacement of mutant sequences will be determined by assaying for normal function of the respective reporter proteins. Experiments will be performed using a variety of parameters to optimize the conditions for SFHR. The parameters to be tested include gene delivery systems (e.g. liposomes), small fragment length, single and double stranded DNA fragments, degree of fragment homology, recombinase proteins and effects of DNA repair proteins.